PROJECT SUMMARY/ABSTRACT Salmonella enterica serovar Typhimurium (STm) cause bacteremia in immunocompromised humans by surviving within the vacuoles of macrophages (M?s). Treatment demands antibiotics that can penetrate to the vacuolar salmonellae and fluoroquinolones are currently preferred. Consequently, resistant STm isolates have emerged and new therapies are necessary. Like for most Gram-negative bacteria, Enterobacteriaceae produce an outer-membrane (OM) lipid bilayer that forms a barrier to the environment and protects against antibiotic killing and host-immune responses. STm regulate the glycerophospholipid (GPL) and the lipopolysaccharide (LPS) molecules within their asymmetric surface bilayer to enhance the barrier and promote survival in M?s during bacteremia. However, the exact bacterial lipid-remodeling proteins and mechanisms necessary during infection are largely unknown. We showed that STm rely upon a conserved enterobacterial transmembrane protein, PbgA, to maintain OM-lipid homeostasis for barrier function and survival in M?s during bacteremia in mice. PbgA binds tetraacylated cardiolipin (CL)-GPL molecules and directs their trafficking to the OM in response to host cues. The data support that STm adapted PbgA to promote LPS stability and OM homeostasis for survival within host vacuoles. We hypothesize that salmonellae exploit structural features and binding interactions of PbgA to control OM-lipid homeostasis during bacteremia. We will pursue this central inquiry by addressing the following specific aims: 1) Define the functional contribution of PbgA-periplasmic domain (PD) subregions and residues for OM-CL trafficking. 2) Define PbgA interactions within the STm envelope necessary for lipid homeostasis and pathogenesis. 3) Delineate the role of PbgA and CL in STm-LPS homeostasis during bacteremia. Minimal knowledge exists for how enteric bacteria transport GPL to the OM. To the best of our knowledge, PbgA is one of the first examples of a protein involved in direct physical translocation of CL molecules across the dual-membrane cell envelope of a Gram-negative bacterium. In understanding PbgA in STm, we hope to inform common mechanisms of OM-lipid trafficking used by enteric pathogens to adapt and survive within the mammalian host environment.